mmg_233_2014_genetics_genomicsfandomcom-20200215-history
Genetically Modified T-cells
CCR5 Gene The CCR5 gene is also known as the human chemokine receptor 5 gene. CCR5 is a cell-surface receptor protein found on the outsides of T-cells, the immune cells responsible for our cell-mediated immunity (3). This gene allows T-cells to communicate with other cells through chemokine-mediated interactions. It is also one of the proteins target by the Human Immunodeficiency Virus (HIV) that allows cell-to-virus interactions to occur. While other proteins found on the surfaces of T-cells are targeted as well, the CCR5 protein is essential for HIV entry into CD4+ T-cells (1; 2). It has been shown that a small percentage of the human population (~1%; this mutation has been observed in ~10% of the European population) contains a mutation in the CCR5 gene, rendering the protein inactive (6). Those born with a homozygous mutation in the gene are entirely resistant to HIV-infection; those born with a heterozygous mutation in the gene may contract HIV, but the progression to AIDS has been shown to be much slower and more easily preventable (2; 3). Scientists have chosen to genetically-modify the CCR5 protein based on evidence of the naturally occurring mutation. Because this genetic mutation occurs naturally in the human population, scientists feel it is the safest genetic-modification option in regards to preventing HIV-infection. In addition, previous literature has shown evidence of a particular patient, known as “The Berlin Patient,” being cured of HIV because of a naturally inherited CCR5 gene mutation (2; 3). To date, he is the only human considered cured of HIV. The Motivation Behind Engineering GM T-cells T-cells possessing the CCR5 gene mutation have a selective survival advantage over all other CD4+ T-cells (4). Scientists have chosen to take advantage of this naturally occurring advantage to improve upon research related to curing HIV. The goals behind this research are vast and bold. Essentially, the idea is to recreate an immune system resistant to HIV-infection (1; 3). By removing hematopoietic stem cells from the blood and bone marrow of an HIV-infected patient, scientists can genetically modify the cells to their liking; these cells can be returned to the body of the patient and function normally, without the risk of HIV-infection (2). The stem cells are harvested from the patient and grown and manipulated in a laboratory. After the genetic modification is final, the population of mutated cells is stimulated to grow to extremely large quantities (around 2 billion cells) (2). When given the same mitogens found in the human body, these stem cells proliferate and differentiate into fully functioning CD4+ T-cells, excluding the newly introduced CCR5 protein mutation. When introduced back into the body of an HIV-infected patient, these cells continue to thrive in their natural environment (3). The Mechanism of Genetic Modification Zinc-finger nucleases (ZFNs) are invaluable molecular tools used to modify eukaryotic genomes (3; 5). A ZFN is essentially a hybrid restriction enzyme; it is composed of highly specific zinc-finger proteins (ZFPs) combined with a bacterial restriction endonuclease, specifically FokI. ZNFs are considered a superior tool for genome editing because of their high specificity for certain DNA recognition sites, as well as their controllable restriction enzymatic activity (3). It is the two major components—the ZFPs and the restriction enzyme—of ZFNs that make them such versatile and useful tools. ZFPs are composed of multiple ‘zinc-fingers’ that provide extremely specific DNA sequence recognition, individually recognizing 3 base pairs within the DNA sequence (5). The FokI restriction enzyme has no sequence specificity, but is highly controllable because it must dimerize before allowing DNA cleavage (3). The goal of using ZFNs in regard to inactivating the CCR5 gene is to allow efficient DNA cleavage while avoiding off-target cleavage events. This can occur because of the dimerization requirement of both ZFNs and the FokI restriction enzyme; these critical components are inactive in their monomer forms (5). To inactivate the CCR5 gene, two ZFNs, composed of highly specific ZFPs, bind simultaneously at ‘half-sites’ on both the left and right sides of the specific DNA sequence that is to be cut. The recognition of this DNA sequence allows the two ZFNs to form a heterodimer, i.e. a dimer composed of two unique ZFNs. The formation of this heterodimer induces dimerization of the FokI restriction enzymes; this second dimerization results in a double-stranded cleavage of the DNA sequence (5). Although the double-stranded cleavage is essential to knocking out the CCR5 gene, it does not actually render the gene inactive. Once the cleavage has occurred, the DNA is repaired through a mechanism called nonhomologous end joining (3). This type of DNA repair allows for many random nucleotide insertions and deletions to occur at the cut site. The permanent disruption of the original DNA sequence renders the CCR5 gene a complete knockout (3). ZFNs have been utilized in the genetic modification of plant, insect, roundworm, and human genes. They possess high potential for future crop engineering, cell line customization, and therapeutic gene correction (5). Success or Failure? The in vivo experiments involving the use of CCR5-deficient T-cells have been successful. An NIAID funded study involving 12 HIV-infected patients tested the feasibility and safety of using genetically modified immune systems to combat HIV-infection. The 12 patients in the trial had their hematopoietic stem cells harvested, genetically modified, stimulated to multiply, and reintroduced into their bodies. The trial members, all of who were undergoing intensive anti-HIV therapy, stopped anti-viral treatment 4 weeks after their stem cell transplants. After 12 weeks of no anti-viral treatments, 10 of the 12 patients had significantly decreased levels of HIV in their systems; one of the 12 patients, with a naturally occurring heterozygous CCR5 gene mutation, had no HIV present in their system (2). References 1) HIV/AIDS Fact Sheet. California Institute for Regenerative Medicine. http://www.cirm.ca.gov/our-progress/hivaids-fact-sheet. Updated 2013. Accessed October 5, 2014. 2) Genetic Modification of Cells Proven Generally Safe as HIV Treatment Strategy. National Institute of Allergy and Infectious Diseases. http://www.niaid.nih.gov/news/newsreleases/2014/Pages/CCR5mutation.aspx. Updated March 5, 2014. Accessed October 5, 2014. 3) Tebas P, Stein D, June CH. Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV. N Engl J Med. March 6, 2014; 370(1): 901-910. doi: 10.1056/NEJMoa1300662 4) Younan P, Kowalski J, Kiem H. Genetic Modification of Hematopoietic Stem Cells as a Therapy for HIV/AIDS. Viruses. November 28, 2013; 5(12): 2946-2962. doi: 10.3390/v5122946 5) Miller JC, Holmes MC, Wang J, et. al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nature Biotechnology. July 1, 2007; 25: 778-785. doi: 10.1038/nbt1319 6) Goedert JJ. Genome Variation among HIV-Resistant People with Hemophilia. NCBI’s Database of Genotypes and Phenotypes: dbGaP. http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000445.v1.p1. Updated October 16, 2012. Accessed October 5, 2014.